Fig. 4.

WT CD4⁺ T cells restore the inflammatory process in αβ TCR KO mice at day 5 post infection. Naïve αβ TCR KO mice were injected via i.p. route with 5 × 10⁷ CD4⁺ T cells isolated by negative selection from spleens of vaccinated WT mice (T cell Trial 4). As controls, mock transfer was performed in age-matched mice in parallel using PBS in identical volume. Five days after adoptive transfer of T cells (or mock transfer with PBS alone), recipient mice were immunized with three doses of SIN/ZPC on day -44, -30, and -16. At 4 weeks following the final vaccination, all animals were challenged i.n. with VEEV ZPC738 (4 × 10⁵ PFU/animal) and monitored daily for disease development and survival. The corresponding survival data is shown in Fig. 3A. Tissues were obtained from randomly preselected animals euthanized on day +5 for organ titration (shown in Fig. 3C), histopathology and immunofluorescence analysis. H&E and immunofluorescent staining of brain sections was performed as described in Materials & Methods. For the latter, sections were incubated with normal goat serum followed by antibody to glial fibrillary acidic protein (rabbit anti-human GFAP, Sigma), or CD3 (T cell marker, rabbit anti-human CD3, DakoCytomation). Alexa 594 conjugated secondary antibody was used for detection. All sections were counter stained with DAPI (Invitrogen) prior to fluorescence microscopy and capture of photographic images (Olympus XL71; DP70 digital camera). VEEV infection results at day 5 post infection are illustrated in H&E images (A to B) and in immunofluorescence (C to D). Three groups of mice were used in this experiment: αβ TCR KO mice transferred with CD4⁺ T cells from WT mice (A and C), and without transfer (B and D). Mice in C did not show a high level of mononuclear infiltration and karyorrhexis as seen in A, in which mononuclear cells defused into the brain parenchyma from vessels of cortex and meninge (arrows). The majority of infiltrated cells are identified as CD3 positive T cells in mice transferred with WT CD4⁺ T cells (arrows in C), and the ring-like stain is a typical feature in staining membrane molecules on T cells which have limited cytoplasm. Mice from the other group only showed few cells with CD3 positivity (arrows in D). Arrowheads in D point to autofluorescence on red blood cells which can be verified by their lack of nucleus.